A late defect in wound healing associated with recurrent spontaneous pneumothoraces: a presumptive role for abnormal collagenase activity.

This case report concerns an individual with a defect in wound healing which resulted in recurring, bilateral pneumothoraces during the late postoperative period. This patient had no history of systemic disease or wound healing abnormalities before his recurrent wound disruption. Physical examination and routine biochemical studies failed to identify any causative agent for the multiple wound dehiscences in the patient. Histologic examination of scar tissue showed collagen fiber bundles with a diameter 50% less than that of normal fibrils. Elastic fibers were barely visible, and the scar tissue included a large number of inflammatory cells. A significant finding was an elevated and aberrant expression of collagenase by a fibroblast cell line established from a skin biopsy specimen. This enhanced level of collagenase expression could be inhibited by treatment of the cells with diphenylhydantoin, an inhibitor of collagenase biosynthesis. After initiation of diphenylhydantoin therapy, the patient's scar formation normalized with the recurrent pneumothoraces. These findings support the conclusion that an abnormal expression of collagenase resulted in enhanced degradation of collagen in the patient's wounds, thereby leading to wound dehiscence.

Hyaluronic acid is a major component of the matrix of fetal rabbit skin and wounds: implications for healing by regeneration.

The extracellular matrix (ECM) of fetal rabbit wounds contains an abundance of the glycosaminoglycan hyaluronic acid (HA) but is devoid of excessive collagen. Thus, fetal wounds heal without scarring, such that tissue repair grossly resembles regeneration. To obtain further insight into the process of fetal wound healing, the ECM of normal fetal rabbit skin was analyzed, thus providing a comparative endpoint for the ECM of healing fetal wounds. Similarities between the matrices would support the theory of healing by regeneration. The glycosaminoglycan (GAG) component of fetal rabbit skin from 24- and 29-day gestational age fetuses was extracted and then quantitated using an alcian blue binding assay. The extracted GAG was characterized by cellulose acetate electrophoresis and HA was identified by its selective digestion by Streptomyces hyaluronidase. The mean GAG content, measured as ng GAG per mg dry weight skin, was 260 +/- 200 for the 24-day group (n = 28) and 280 +/- 220 for the 29-day group (n = 26). The only GAG identified at both times of gestation was HA. This study has demonstrated that HA is the predominant GAG present in fetal rabbit skin and its quantity is stable during the period studied late in gestation. A major component of the ECM of both wounded and normal fetal skin is HA, indicating a close compositional similarity. These observations provide biochemical support for the hypothesis that the reparative process of injured tissue in the fetal rabbit proceeds in an attempt to reconstitute normality, i.e. regeneration.

Analysis of skin grafting techniques in the fetal rabbit.

The experimental model reported here was developed initially to examine the possibility of in utero coverage of congenital soft tissue defects using several types of reconstructive techniques. To pursue this, full-thickness skin grafts, pedicle flaps, and skin "islands" were fashioned on the backs of fetal rabbits; equivalent adult control wounds were also created. While all pedicle flaps and skin islands remained viable, none of the full-thickness grafts survived in the fetus. All adult control flaps, skin islands, and skin grafts were viable. Angiogenesis is crucial to full-thickness skin graft survival. These observations suggest that the death of full-thickness fetal skin grafts may be related to a failure of neovascularization in the graft bed. Further analysis using this model may help elucidate the factors involved in fetal angiogenesis. Additionally, this model may permit testing of putative angiogenic factors applied under a full-thickness skin graft; graft survival offers an easy, objective, and quantifiable means of data analysis.

A microassay to quantitate collagen synthesis by cells in culture.

A method to quantitate collagen synthesis, total protein synthesis, and DNA in 24-well culture plates is presented. Collagen-producing cells such as human intestinal smooth muscle cells and dermal fibroblasts were pulse-labeled with [3H]proline. After incubation, the plates were heated to 90 degrees C to stop isotope incorporation and sonicated to lyse the cells and an aliquot was removed for DNA quantitation. Carrier protein was added, all protein was precipitated by trichloroacetic acid, and unbound isotope was removed by repeated precipitations. After incubation with purified bacterial collagenase, both the soluble 3H-labeled collagen-derived peptides and the remaining insoluble 3H-labeled noncollagen protein were quantified. Results were expressed as the amount of radioactivity incorporated into collagen and noncollagen protein per nanogram DNA and also as the percentage of collagen synthesis per total protein synthesized. The advantage of this technique over previous attempts to scale down the assay is that the entire assay for DNA, collagen, and non-collagen protein can be carried out in the same well without any transfer of material. This technique also provides a significant savings of culture medium, serum, growth factors, and cell material.

Fetal skin organ culture.

The fetal response to cutaneous injury has been investigated in a variety of models; most have studied the differences between fetal and adult healing mechanisms in vivo and in cell culture. Further disclosure of the cellular and biochemical events requires a model that can be manipulated to study single factors influencing fetal tissue repair without the complex interactions that occur in vivo, but in a system that more closely approximates normal skin than cell culture models. This paper presents a method for the organ culture of fetal skin and its advantages as a model to help elucidate fetal healing mechanisms. Skin sections (1 x 1 cm) excised from the backs of fetuses of New Zealand white rabbits on day 27 gestation (term = 31 days) were placed eccentrically in 65-mm culture dishes and fed daily with 2.5 mL of DMEM containing 10% fetal calf serum, antibiotics, and 10 mM ascorbic acid. A separate group, treated similarly, received 4-mm punch wounds to assess the in vitro response to wounding. The specimens were incubated at 37 degrees C in humidified room air on a rocker platform to provide alternate exposure of the skin to air and medium. Gross observation at 3 weeks showed cells extending into the central wound, indicating that viable cells were proliferating and/or migrating from the tissue. Skin was examined histologically and was viable over the 3-week period studied. Organ culture, by maintaining tissue in the natural extracellular matrix, allows cell-to-cell contact and communication to be maintained while allowing controlled environmental manipulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Operative techniques in the fetal rabbit.

The development of fetal surgical techniques has made the antenatal correction of congenital defects possible. These techniques have evolved from trials with animal models, permitting increasingly sophisticated operations with low morbidity and mortality. Experimental models range from large animals offering longer gestations but with single pregnancies and high cost, to smaller animals offering multiple pregnancies at reduced cost but with shorter gestations. This paper describes operative techniques in the fetal rabbit and its advantages as a fetal surgical model. Experience with the pregnant rabbit has shown it to be a suitable surgical model for several reasons. Pregnancies are multiple, increasing cost effectiveness and permitting operation on up to eight fetuses per litter without fetal loss. Techniques that promote fetal survival include local housing of does several days prior to operation and preoperative sedation. Spontaneous mask ventilation provides ease of anesthetic administration and titration. Overall surgery is well tolerated with a low incidence of intraoperative complications. Rabbit models have been used in the study of transamniotic fetal feeding, abdominal wall defects, and wound healing. These techniques have resulted in postoperative fetal viability approaching 90%, with negligible maternal mortality in over 4000 fetal operations, thereby making the rabbit a manageable cost-effective model of fetal surgery.

Establishment of an adult rat fibroblast cell line for studies of collagenase regulation.

Several dermal fibroblast lines have been established from explants taken from adult rats. The cells have been cultured for 2 yr and possess stable and well-defined growth characteristics through subculture 18. The cells are readily stored in liquid nitrogen with good viability after thawing. Collagenase activity secreted into the culture medium of the cells at different periods of growth has been examined. There is an 88% drop in total enzyme activity present in the medium between 4 and 14 d of culture, when the cells were plated to reach confluence at Day 8 to 10. A more pronounced fall is noted at earlier times when the cells are plated at a higher density. The correlation between DNA content of the cell monolayer and enzyme activity was -0.895, indicating a possible relationship between the growth of cells and collagenase release.